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Journal of Geophysical Research: Biogeosciences

RESEARCH ARTICLE
10.1002/2013JG002598
Key Points:
Measured changes in uorescence of
three DOM sources over pH gradients
Combined parallel factor analysis and
self-organizing maps
Differences in uorescence composition
evident over small pH ranges

Supporting Information:
Figures S1-S2 and Table S1
Text S1
Text S2
Correspondence to:
C. Guguen,
celinegueguen@trentu.ca

Citation:
Cuss, C. W., Y. X. Shi, S. M. McConnell,
and C. Guguen (2014), Changes in the
uorescence composition of multiple
DOM sources over pH gradients
assessed by combining parallel factor
analysis and self-organizing maps,
J. Geophys. Res. Biogeosci., 119,
doi:10.1002/2013JG002598.
Received 19 DEC 2013
Accepted 21 AUG 2014
Accepted article online 23 AUG 2014

Changes in the uorescence composition of multiple

DOM sources over pH gradients assessed
by combining parallel factor analysis
and self-organizing maps
C. W. Cuss1, Y. X. Shi2, S. M. McConnell3, and C. Guguen2
1

Environmental and Life Sciences Graduate Program, Trent University, Peterborough, Ontario, Canada, 2Chemistry
Department, Trent University, Ontario, Canada, 3Department of Computing and Information Systems, Trent University,
Peterborough, Ontario, Canada

Abstract

Dissolved organic matter is a ubiquitous constituent of natural waters that plays key roles in
several important processes. The uorescence properties of DOM have been linked to its functionality, but
these properties may vary with pH. In this study Kohonens self-organizing maps (SOMs) were applied to
excitation-emission matrices (EEMs) of fresh dissolved organic matter (DOM) from three sources: senescent
sugar-maple leaves and white spruce needles, and humied white spruce needles, over a pH range of ~4.5 12.5.
SOMs were applied to raw EEMs, EEMs reduced in dimensionality by pre-processing using parallel factor
analysis (PARAFAC), and PARAFAC loading proportions normalized to values at initial pH. Some separation of
EEMs into source-based clusters was achieved in the SOM of raw EEMs, but commingling was apparent and
evidence of changes over pH gradients was overshadowed. SOMs of PARAFAC component proportions
demonstrated clear source-based clustering, and pH-based gradients were visible for DOM from senescent and
humied spruce needles. Changes in optical properties were obvious over pH gradients in the SOM of
components normalized to starting condition. Component proportions decreased to values as low as 5% of the
initial values for microbial humic-like peak M and increased to as high as 278% for a humic-like component.
Tyrosine-like uorescence increased to 112% of initial over increasing pH in humied spruce leachates
but decreased to as low as 45% in the other leachates. The combination of PARAFAC and SOM drastically
enhanced visualization and interpretability of pH-induced changes in DOM compared to either method alone.

1. Introduction
Dissolved organic matter (DOM) is a dynamically polymorphous mixture of molecules that is ubiquitous in
natural waters and functions in many important roles, such as providing a source of nutrients, energy, and
shelter for microorganisms [Zepp et al., 2006; Fellman et al., 2008]; governing the toxicity, mobility, and
speciation of heavy metals [Stevenson, 1994; Luster et al., 1996; Guguen and Dominik, 2003], and forming
carcinogenic disinfection byproducts (DBPs) during water treatment [Beggs and Summers, 2011].
Fluorescence spectroscopy, and particularly excitation-emission matrices (EEMs)[Coble, 1996], has been
broadly used to connect the optical properties of DOM to its functionality [Andrade-Eiroa et al., 2013].
However, the uorescence properties and structure of DOM are sensitive to pH changes [Patel-Sorrentino
et al., 2002; Spencer et al., 2007; Yi et al., 2009], so that the connection of DOM to its functionality under varying
pH conditions requires that the relationship between DOM uorescence and pH is well understood.
Kohonens self-organizing maps (SOMs) [Kohonen, 2001] are single-layer articial neural networks originally
developed for use in articial intelligence, which have recently been introduced in environmental science
[Astel et al., 2007; Lhr et al., 2010; Roig et al., 2011], and have been used to assess the functionality of full
uorescence EEMs [Bieroza et al., 2009, 2012a]. However, SOMs of full EEMs are limited in their ability to
identify specic uorophores, which restricts their applicability for linking uorescence to functionality.
Further, the distance measures commonly used in SOMs (e.g., Euclidean distance) are such that the large
number of excitation-emission (Ex/Em) pairs in EEMs (thousands to tens of thousands) presents a secondary
challenge for the simultaneous evaluation of multiple variables from different measurements (e.g., extremely
high weightings), and local maxima may result in sub-optimal ts. The connection of EEM uorescence to
DOM functionality requires the simultaneous measurement of multiple variables in addition to EEMs, where

CUSS ET AL.

2014. American Geophysical Union. All Rights Reserved.

Journal of Geophysical Research: Biogeosciences

10.1002/2013JG002598

the relationships between these variables and their distributions may be complex and uncertain.
Thus, it would be benecial to reduce the dimensionality of the information embedded in EEMs prior
to applying SOMs.
Parallel factor analysis (PARAFAC) [Stedmon et al., 2003; Stedmon and Bro, 2008] has been instrumental in
reducing the dimensionality of EEMs to a manageable number of independent, semi-quantitative
variables (often < 10). PARAFAC uses least squares error minimization to optimize estimates of
independent, underlying uorophore groups in EEMs through the tri-linear regression of correlated
excitation-emission wavelength pairs [Stedmon and Bro, 2008]. PARAFAC components have also been
directly related to functional effectiveness as regards nutritional value [Fellman et al., 2008; Cuss and
Guguen, 2012], metal-binding properties [Yamashita and Jaff, 2008; Cuss and Guguen, 2012], DBPformation potentials [Beggs and Summers, 2011], and DOM removal efciency in water treatment
[Bieroza et al., 2012a]. PARAFAC compared well with SOMs when applied to EEMs for assessing the
effectiveness of DOM removal during water treatment [Bieroza et al., 2012a]. However, SOMs also offer
several advantages over PARAFAC, such as excellent data visualization capabilities and ease of
interpretation, the ability to handle noisy data with complex, unknown (inter)relationships between
several variables, and the ability to model small data sets [Kohonen, 2001; Bieroza et al., 2009, 2012a].
Thus, reducing the dimensionality of EEMs by pre-processing using PARAFAC may improve the SOMs of
uorescence signatures and increase its applicability by facilitating combined analysis with other
variables. In the case of pH-motivated changes in the uorescence composition of multiple DOM
sources, the relationships between variables are especially complex (i.e., log scale compared to
compositional data with differing initial conditions). Importantly, applying SOM to PARAFAC component
loadings expressed as proportional uorescence facilitates a more holistic picture of DOM that accounts
for its overall uorescence composition. Given that DOM is a complex mixture of constituents, this
compositional mode provides a new perspective on changes to the entire uorescence signature,
rather than inspecting isolated uorophores.
In this study, the uorescence of several different sources of fresh DOM (i.e., leaf leachates) was assessed over
pH gradients by combining PARAFAC and SOMs. Results were compared to those obtained using only SOMs
on full EEMs, and the merits of both approaches were compared.

2. Materials and Methods

2.1. Sampling, pH Titrations, and Instrumental Analyses
Leaf litter was collected from three sites at the Trent University campus in Peterborough, Ontario, Canada
(44.36N, 78.29W): freshly fallen sugar maple leaves (Acer saccharum), senescent needles stripped from white
spruce (Picea glauca), and aged, multi-year needles collected from beneath a second white spruce. Litter was
air-dried for 24 h, frozen at 5C to simulate winter conditions and thawed prior to leaching in the dark
following a previously developed method [Cuss and Guguen, 2012]. Briey, litter was placed in precombusted (450C for 5 h) glass beakers and immersed in 200 mL of Milli-Q water (MQW, 18 M cm 1;
Millipore). Leachates were decanted, discarded, and replaced with MQW 1and 3 h after immersion, and the
5 h leachate was used for analysis. Samples were ltered sequentially through combusted, rinsed, 0.7 m
lters to isolate DOM (GF/F, Whatman), and lter-sterilized (0.22 m nitrocellulose; Millipore). Ten distinct
leachates were generated using sub-samples of the litter: four for maple (M1M4), three for senescent spruce
(S1S3), and four for aged (old) spruce (O1O4).
Aliquots of NaOH solution (Sigma-Aldrich) were added to each leachate while stirring, and the pH was
measured using an Accumet portable pH meter. Solution pH was allowed to achieve a constant value
(~2 4 min.) before two 5 mL aliquots were sampled for absorbance and uorescence analyses. For each
leachate, three sets of three to four aliquots were taken at 11 different pH values spanning a range from
~ 4.5 to 12. Leachates were stored in the dark for at 4C between sets (~1.5 h was required for uorescence
analysis). After removal from storage, leachates were warmed to room temperature, and the pH was
measured. A slight reduction in pH (0.7 0.2, n = 20) was observed compared to the value prior to storage,
presumably due to hysteresis effects. In selected experiments, HCl was also added to reduce the pH prior to
adding NaOH (S1 and O1) and to return the pH to its initial value after titrating with NaOH (S3). The pH ranges
for each experiment are summarized in Table 1.

CUSS ET AL.

2014. American Geophysical Union. All Rights Reserved.

Journal of Geophysical Research: Biogeosciences

10.1002/2013JG002598

Table 1. Range of pH Values for Titrations in Each Experiment

Series of pH Changes
Experiment
Maple 1 (M1)
Maple 2
Maple 3
Maple 4
Spruce 1 (S1)
Spruce 2
Spruce 3
Old spruce 1 (O1)
Old spruce 2
Old spruce 3
Old spruce 4
a

Initial

10

4.4
4.5
4.9
4.4
5.2
5.6
5.4
6.4
6.1
6.3
6.0

4.6
6.5
5.9
5.9
4.5
8.9
8.9
2.9
6.7
9.1
9.0

4.9
6.8
6.2
7.8
5.0
9.1
9.5
5.4
7.0
9.5
6.5

4.8
7.1
6.4
7.9
8.5
9.6
10.2
5.7
7.1
8.2
9.8

5.0
7.4
6.4
8.5
9.0
10.0
10.7
6.1
7.5
10.0
10.2

5.2
7.8
6.6
9.1
9.6
10.5
11.2
6.8
7.8
10.3
10.6

5.8
8.0
7.0
9.3
10.1
10.9
11.6
8.1
8.6
10.6
10.9

6.0
8.1
7.2
8.9
10.5
11.3
11.8
7.0
8.0
10.9
10.9

7.7
8.3
7.9
9.2
11.0
11.6
12.1
7.8
9.0
11.2
11.3

7.9
8.6
8.5
9.6
11.5
12.0
9.3
8.4
9.9
11.6
11.5

9.2
9.0
9.0
10.2
12.0
12.3
5.5
9.3
11.0
12.0
12.0

Instances when acid was added are underscored.

The absorbance spectrum was measured over a range of 250 700 nm in 1 nm increments (Shimadzu UV-2550).
When necessary, samples were diluted using MQW adjusted to the same pH so that the absorbance at
250 nm was < 0.05 AU to minimize the inner-ltering effect [Lakowicz, 2006]. The uorescence
excitation-emission spectrum (EEM) was measured by scanning its emission (Em) over the wavelength
range of 300 600 nm for excitation (Ex) wavelengths of 250 500 nm, both in increments of 5 nm in
ratio (S/R) mode (Horiba Jobin-YvonFluoromax 4). EEMs were corrected using the manufacturer-generated
emission and excitation correction factors and calibrated to Raman units using the area under the Raman
scatter peak of MQW [Lawaetz and Stedmon, 2009]. Background scatter was removed by subtracting an EEM of
ultrapure Milli-Q water (MQW) measured daily.
2.2. Data Analysis
2.2.1. Parallel Factor Analysis (PARAFAC)
The PARAFAC algorithm uses alternating least squares to minimize error during tri-linear regression of
independent groups of correlated excitation-emission wavelength pairs (i.e., factors or components). As part
of this tri-linear regression, factor loadings are estimated for all components in each sample, where these
loadings are proportional to concentration. Thus, PARAFAC provides a semi-quantitative measure of DOM
composition in terms of its independent uorophores. Parallel factor analysis was conducted on 121 EEMs
using Matlab R2010a, following the guidelines outlined in the DOMFluor toolbox [Stedmon and Bro, 2008].
Aliquot M4-9 (pH 9.6) was identied as an outlier and removed due to aberrant uorescence. The nal model
was validated using split-half analysis and the Tucker congruence coefcient with random initialization
[Stedmon and Bro, 2008].
The proportion of total uorescence owing to each PARAFAC component was calculated by dividing the loading
of each component (Ci) into the total of the loadings within each aliquot (i.e., Ci/Ci for all i PARAFAC components).
Variation in uorescence caused by changing the pH was isolated from variation caused by differences in
leachate properties by normalizing the component loadings to their initial values before the pH was changed.
That is, the loading of each PARAFAC component in each aliquot (A0 A10) was divided by the loading of the
corresponding component in the initial aliquot A0 of the same experiment (i.e., [proportion of component in
aliquot Aj] / [proportion of component in aliquot A0] for j = 0 10). In three cases the PARAFAC component
loadings were 0 in aliquot A0 but increased with increasing pH (C6 and C7 in experiment S3 and C4 in
experiment O4), preventing transformation to proportions in all subsequent aliquots. Consequently, proportions
were left as 0 in A0 to distinguish the aliquots, while the average loadings for the respective components in A0
from the other experiments with the same source were used to calculate the normalized change in A1A10.
2.2.2. Kohonens Self-Organizing Map (SOM)
Kohonens self-organizing map (SOM) arranges multidimensional data across gradients of similarity through the
iterative adjustment of variables in neurons, resulting in a distribution that asymptotically approaches that of
the input data [Kohonen, 2001]. The map is organized on a two-dimensional lattice, where each node represents
a single neuron, and each neuron is represented by a vector of variables with dimensionality equal to that of the
input samples (i.e., codebook vector). Neurons are numbered in increasing order from top to bottom, left to

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2014. American Geophysical Union. All Rights Reserved.

Journal of Geophysical Research: Biogeosciences

10.1002/2013JG002598

Figure 1. Parallel factor analysis (PARAFAC) components detected in this study. Naming follows Coble [1996].

right (e.g., Figure S1 in the supporting information). For further details about the testing used to optimize map
parameters in this study, and the operation and mathematical foundations of SOMs, the interested reader is
directed to the supplementary information and the comprehensive work of their originator [Kohonen, 2001].
The same 120 raw sample EEMs analyzed by PARAFAC were also analyzed by SOM using the SOM Toolbox in
Matlab R2010a, following the procedures outlined in Bieroza et al. [2012b]. Conveniently, EEM pre-processing
in the tutorial of Bieroza et al. [2012b] follows the same procedure as that used in the DOMFluor toolbox,
so that combination and comparison of PARAFAC and SOMs are relatively simple. The SOM algorithm was also
applied to component proportions in order to assess the extent to which useful information was lost by the
PARAFAC algorithm. Component proportions were chosen over raw loadings because they remove differences
caused by variable DOC concentration in the different experiments and result in a distribution of variation that
is better explained by the rst two latent variables (see discussion under the heading of Map quality
testing in Text S2 in the supporting information). Differences in optical properties were also limited to
pH-associated changes by applying the SOM algorithm to proportions of PARAFAC components
normalized to their respective values in the original aliquots (A0), in which the pH was unaltered.

3. Results and Discussion

3.1. Parallel Factor Analysis
Seven components were successfully split-half validated using PARAFAC, explaining 99.8% of total variation
(Figure 1). Component 1 (C1) had an excitation/emission maximum (Ex/Em) of 275/305 nm and was similar
to the protein-like B peak [Coble, 1996], which has also been associated with polyphenols such as tannins
[Maie et al., 2007], and with leaf leachates [Beggs and Summers, 2011; Cuss and Guguen, 2012, 2013].
Components C3 (Ex/Em = 280/325 nm) and C4 (Ex/Em = 255/350 nm) were also protein/polyphenol-like,
where the latter resembled tryptophan-like uorescence [Coble, 1996]. Component C2 (Ex/Em = 305/440 nm)
was similar to microbial humic-like uorescence, while C5 (Ex/Em = 340/450 nm) resembled humic-like.
C6 (Ex/Em = 255(295)/425 nm) and C7 (Ex/Em = 270(370)/525 nm) were similar to fulvic/humic-like
components previously identied in rivers and wetlands [Stedmon et al., 2003; Fellman et al., 2008], marine
systems [Coble, 1996; Stedmon et al., 2003; Maie et al., 2007], waste waters [Saadi et al., 2006], and leaf
leachates [Luster et al., 1996; Beggs and Summers, 2011; Cuss and Guguen, 2012, 2013].
3.2. Kohonen Map of Full EEMs
The SOM U-matrix (Figure 2A) shows the distance between each node of the map and its neighbors. Larger
distances (i.e., less similarity) are shown in red on the U-matrix, demonstrating that the EEMs of old spruce

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Journal of Geophysical Research: Biogeosciences

10.1002/2013JG002598

Figure 2. (A) U-matrix, (B) hit histogram, and (CE) mean sample pH values for nodes of self-organizing map (SOM) uorescence as full excitation-emission matrices
(EEMs) for the leachates of old spruce (green), senescent spruce (blue), and senescent maple (red). pH values for nodes containing EEMs from HCl-modied
experiments are shown in black. The amount of coloring on the hit histogram is proportional to the number of associated EEMs.

were very different from the other samples. The hit histogram (Figure 2B) denotes the number of EEMs that
were the most similar to each map node, which is also proportional to the amount of color added. Clusters of
samples are identied by their distance from other samples. Each cluster was primarily associated with a
single leachate, and the senescent and old spruce leachates were each split into two clusters (Figure 2B).
Further, aliquots from all experiments were spread across the map along pH gradients (Figures 2C2E).
Experiments in which HCl was added (Old spruce 1 and Spruce 1, 3; Table 1) displayed different behavior over
similar pH ranges compared to the other experiments. For example, experiment O1 comprised a cluster that was
separate from the other old spruce leachates (lower right corner, Figure 2C). Additionally, the aliquots for
experiment S1 were spread along the x axis over a relatively short gradient with increasing pH, while those for
S2 and S3 migrated over two large steps and distinct clusters in the x and y axes (e.g., N1-N5-N49; Figure 2D).
Finally, aliquot S3-10 (returned to pH 5.5 from 12.1 using HCl) shared the same neuron as S3-1 (pH 8.9, N20)
rather than the neuron with a more similar pH (N1, aliquot S3-0, pH 5.4). These differences strongly suggest that
pH-related changes to the optical properties of leachate DOM exhibit path dependence and so are not
necessarily reversible. Such pronounced irreversibility could be caused by the mixture of reactive polyphenols,
tannins, proteins, and carbohydrates that are produced during leaching. For example, proteintannin complexes
form quite readily in leachates and are affected by pH [Kraus et al., 2003]. This suggests that changes in pH
should be minimized during the processing of leaf leachates and DOM that may contain fresh material if their
natural optical and conformational properties are of interest.
To illustrate changes in uorescence associated with increasing pH, the codebook vectors are shown for
key neurons along gradients of change (Figure 3). Changes in the codebook vectors along paths of
increasing pH for leachates demonstrated that increasing pH resulted in a general trend toward greater
amounts of microbial humic-like and humic/fulvic-like uorescence, and less protein-like uorescence.
However, it was difcult to distinguish trends in proximate components purely on the basis of codebook
vector EEMs alone (e.g., humic-like C2, C5, and C6 or protein-like C1, C3, and C4; Figures 1 and 3). Signicant
increases in humic- and fulvic-like uorescence have been previously noted in the DOM isolated from
CUSS ET AL.

2014. American Geophysical Union. All Rights Reserved.

Journal of Geophysical Research: Biogeosciences

10.1002/2013JG002598

Figure 3. Selected codebook vectors for neurons in SOM of full EEMs numbered by neuron and associated pH (Figure 1).
Neurons are numbered top to bottom, left to right on the SOM (e.g., Figure S1 in the supporting information). Arrows
demonstrate changes observed with increasing pH for A) old spruce, B) senescent spruce, and C) maple.

headwaters over a pH range of 2 10 [Spencer et al., 2007], and from Amazon basin rivers over a pH range
from 2 to 12 [Patel-Sorrentino et al., 2002].
3.3. SOM of Component Proportions
Distinguishing the behavior of specic uorophores could be improved by inspecting the component
planes, which show the distributions of individual variables over the full map. However, this
interpretative method is far from convenient for codebook vectors composed of EEMs, in which the set
of variables is dened by thousands to tens of thousands of Ex/Em pairs. Since PARAFAC provides a
single variable for each uorophore and has also been useful for connecting distinct uorophores to
their functional effectiveness (e.g., in metal-binding and DBP formation) [Luster et al., 1996; Yamashita
and Jaff, 2008; Beggs and Summers, 2011; Cuss and Guguen, 2012; Chen et al., 2013], the viability of
performing a SOM of the PARAFAC component proportions was investigated (Figure 4).
The clustering of samples according to DOM source for the SOM of component proportions was very
similar to that of the raw EEMs, but there was no mixing of leachates from different sources (i.e.,
compare Figures 2B and 4A). On the other hand, the structure of the source-based clusters was more
separated by experiment for the SOM of proportions, making change less evident over the pH gradient

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2014. American Geophysical Union. All Rights Reserved.

Journal of Geophysical Research: Biogeosciences

10.1002/2013JG002598

Figure 4. (A) Hit histogram, (B) mean pH values, and (C) component planes for SOM of PARAFAC component proportions for the leachates of old spruce (green),
senescent white spruce (blue), and senescent sugar maple (red). The amount of coloring on the hit histogram is proportional to the number of associated EEMs.

for leachates that experienced more subtle changes with pH (i.e., maple and senescent spruce;
Figures 2D, 2E, and 4B). The transformation to proportional uorescence forces the variables of each
sample into a correlated conguration (seven-dimensional simplex; Figure 4C). In this case, the
component planes describe changes in the proportion of each PARAFAC component over the SOM. This
format is readily interpreted, from which it can be seen that the primary differences between the
leachates can be expressed in terms of the relative proportions of PARAFAC components C1, C2, C3, and
C7: the proportion of C2 is higher for maple leachates, C1 and C3 for senescent spruce, and C7 for old
spruce. This result agrees with previous ndings for leaf leachates, in which spruce leachates were
dominated by protein-like uorescence, maple by microbial humic-like, and older, over-wintered leaves
had greater amounts of humic-like material [Cuss and Guguen, 2013]. Despite sacricing some clarity
insofar as changes over pH gradients was concerned, the SOMs of PARAFAC proportions and loadings
offered several advantages over that for the full EEMs: (1) a much smaller number of readily
decipherable component planes, allowing easy interpretation of variation in optical properties over the
map; (2) a much lower computation time (seconds compared to hours in this study, where most
computation time was spent initializing the map); and (3) eases combination with other variables
without the need for heavy weightings to compensate for the bias in the distance measure caused by
thousands of Ex/Em pairs.
3.4. SOM of Change in Component Proportions
To focus on changes with pH, the SOM was applied using PARAFAC component proportions (Figures 5 and 6).
Since all component proportions were normalized to the starting condition, all sources were initially
associated with the same neuron (N3) and then moved across the map as pH changed. Hence, differences
in direction from N3 represented different changes in the proportions of PARAFAC components,
demonstrating different structural changes.

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2014. American Geophysical Union. All Rights Reserved.

Journal of Geophysical Research: Biogeosciences

10.1002/2013JG002598

Figure 5. Hit histogram with increasing pH-related changes illustrated by arrows for leachates of: (A) old spruce (green), (B) senescent white spruce (blue), and (C)
senescent sugar maple (red). Dashed arrows show changes observed with increasing pH (solid for experiments with HCl added). The amount of coloring on the hit
histogram is proportional to the number of associated EEMs.

Changes in optical properties along pH gradients clearly differed by source, as revealed by comparing the
paths of each leachate type (Figures 5A5C) with the corresponding changes in component proportions on
the component planes (Figure 6). For example, EEMs generally moved from left to right (old spruce, O1
excepted) and from top to bottom (senescent spruce, S1 excepted) across the map as they increased in pH.
These pH changes corresponded with a small increase in the proportion of protein-like C1 for the old spruceneedle leachates (mean SD increased from 27.8 1.6 to 29.8 1.2%), and a drastic decrease in the
proportion of C1 for the leachates of senescent spruce needles (from 76.4 2.0 down to 33.1 0.0%).
Meanwhile, the proportion of humic/fulvic-like C6 remained relatively unchanged in the leachates of old
spruce needles (21.6 0.8 at start vs. 21.2 0.2%) and increased up to 19-fold in the leachates of senescent
spruce needles (from 0.3 0.5 up to 13.9 5.6%). Hence, the proportion of humic-like uorescence in humicdominated, older spruce-needle leachates did not change considerably while the humic-like uorescence of
the protein-dominated senescent spruce-needle leachates increased substantially. In other words, the same
PARAFAC component behaved differently in the two DOM sources, despite being exposed to the same
treatment. These changes were also evident in the raw PARAFAC loadings (Fmax; data not shown) and so
were not an artefact of proportionality.
Leachate-based differences were also observed for the proportion of microbial humic-like C2, which
increased slightly in maple-leachate DOM where it was initially the highest (16.0 2.5 to 17.3 2.7%), and in
the leachates of senescent needles (2.5 0.2 to 4.5 2.3), but decreased to negligible levels (1.2 0.2 to
0.0 0.0%) in the DOM from old needles despite starting at relatively low levels in both needle leachates.
The proportion of C1 in experiment O1 increased more (22.8 to 28.0%) than in O24 (27.8 1.6 to 29.8 1.0%)
despite its lower maximum pH (9.3 compared to 11, 12, and 12 for O24, respectively). The pH of O1 was
reduced to ~3 at the beginning of this experiment (Table 1 and Figure 5A), suggesting that this difference
may have been caused by irreversible conformational changes. Similar differences were observed for
experiment S1 which was initially reduced to pH 4.5, with the consequence that less increase was observed in
the proportions of C2, C3, and C57 compared to the other leachates of senescent spruce needles (e.g.,
compare component proportions at N53 on Figure 6 to those along the bottom row).

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2014. American Geophysical Union. All Rights Reserved.

Journal of Geophysical Research: Biogeosciences

10.1002/2013JG002598

Figure 6. Component planes from SOM of PARAFAC component proportions normalized to initial condition.

The increase in the proportion of C5 was marked in the leachates of senescent spruce needles, up to
278 times its initial proportion (0.3 0.3 to 14.1 0.7%). Interestingly, after this sharp increase the
proportions of humic/fulvic-like components C5C7 then declined slightly at pH > ~11.5 in the spruce
leachates (Neurons 11, 22, and 33), which has been previously noted for DOM isolated from rivers of the
Amazon basin [Patel-Sorrentino et al., 2002].
The leachates of sugar maple leaves exhibited the least change in their PARAFAC composition, with a
slight decrease in the proportion of C1, and slight increases in the proportions of C5 and C7, similar to the
leachates of senescent spruce needles (Figures 5C and 6). The difference in the magnitude of change may
have been due to the lower maximum pH for maple compared to that of spruce (~10 compared to 12;
Table 1). However, the composition of leachates from deciduous leaves and coniferous needles differs
substantially [Kuiters and Sarnik, 1986; Kraus et al., 2003], suggesting that the changing pH may have
provoked different compositional changes for the different DOM sources.
The formation of protein-polyphenol complexes occurs both in leaves during senescence and in soils where pH
exerts considerable inuence [Httenschwiler and Vitousek, 2000; Kraus et al., 2003]. However, the hydrogen
bonding and structure of plant phenolics become unstable at high pH [Friedman and Jrgens, 2000], likely
affecting the nature of bonds between polyphenols and proteins. Since the uorescence signature of proteins is
highly sensitive to binding with other molecules [Vivian and Callis, 2001], the increase in protein-like C3 and
corresponding decrease in tyrosine-like C1 observed for spruce leachates may indicate changes in the DOM
structure caused by changing interactions between polyphenols and proteins [e.g., red shift induced by

CUSS ET AL.

2014. American Geophysical Union. All Rights Reserved.

Journal of Geophysical Research: Biogeosciences

10.1002/2013JG002598

hydrogen bonding of tyrosine; Lee and Ross, 1998]. Similarly, the increase in tryptophan-like uorescence
observed in old, more humied spruce leachates may stem from the release of small amounts of
proteins that had been encapsulated by humic substances [Henderson et al., 2009; Fellman et al., 2010;
Tomaszewski et al., 2011].
While these explanations remain speculative in the absence of further testing, it is clear that the coupling of
PARAFAC and SOM is capable of revealing differences in the transformations of DOM leached from leaf litter.
The SOM of the full EEMs provided easily interpretable uorescence signatures (neuron EEMs) for the
different sources. The SOM of PARAFAC component proportions readily revealed the differences in
uorescence composition between sources. However, the large differences between sources masked the
different responses of the sources when the pH was changed, which was exposed by rst normalizing the
proportions to starting condition. The ability of self-organizing maps to organize data with uncertain
distributions readily facilitated this data transformation, enabling the visualization of organization across
multiple modes of variation (i.e., differences in composition and pH response). Since this coupling provides a
variety of modes for readily visualizing DOM properties, it may prove to be highly valuable for investigating
the biogeochemical transformations of DOM. The descriptive capability of this coupling may also be
enhanced by adding other indicators of DOM structure and composition to the SOM variable set, providing a
more robust description of the changes in overall DOM composition.

4. Conclusions and Implications

The combination of SOM and PARAFAC was found to be a powerful tool for interpreting relationships
between optical properties both between distinct DOM sources, and over pH gradients. Given the ease of
implementation using the SOM toolbox, and the power of the combined analysis, the authors highly
recommend combining PARAFAC and SOM analyses in future studies. This combination will be useful both for
linking changes in specic PARAFAC components to their function through the simultaneous measurement
of other variables, and for readily visualizing higher-order relationships of unknown natures between
these variables.
The uorescence compositions of DOM isolated from three leaf leachates changed signicantly in response
to increasing pH, and these changes exhibited striking differences by source: leachates from more humied,
multi-year spruce needles tended to increase in tyrosine-and tryptophan-like uorescence and decrease
in microbial humic-like uorescence, while the leachates of senescent maple leaves and spruce needles
tended to increase in protein/polyphenol-like and humic-like uorescence while decreasing in tyrosine-like
uorescence. These differences demonstrate that the uorescence of PARAFAC components in DOM from
different sources does not necessarily behave similarly under identical treatments. Since DOM uorescence
may exhibit source-dependent responses to the same experimental treatment, the uorescence of individual
PARAFAC components may not be a reliable indicator of DOM functioning. Hence, efforts seeking to connect
the uorescence of DOM to its functionality should consider the complete uorescence composition
rather than relying upon the responses of individual PARAFAC components.

Acknowledgments
The authors thank A. McDonough for
species identication. This work was
funded in part by the Canada Research
Chairs program (CG), the Canadian
Foundation for Innovation (CG), and the
Canadian National Science and
Engineering Research Council (CG).
CWC gratefully acknowledges the
nancial support accorded by the
Alexander Graham Bell Canada
Graduate Scholarship.

CUSS ET AL.

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